This investigation is a continuation of studies of the subunit-subunit interactions of the biotin enzyme, transcarboxylase. The enzyme is made up of a hexameric central subunit which, in electron micrographs, appears to have a cylindrical shape. Six dimeric outer subunits are attached at the opposite faces of this central subunit, 3 at each face. The linkage of the outer to the central subunit is via 12 biotinyl subunits, 2 per outer dimeric subunit. The reaction catalyzed is CH3CH(COO-)COSCoA plus CH3COCOO- yield. The CoA ester sites are on the central subunit and the keto acid sites on the outer subunits and the biotin serves as a carboxyl carrier between these sites. On the central subunit, there are 12 sites through which the outer subunits are attached, via a portion of the biotinyl subunit, 12 sites for binding of another part of the biotinyl subunit for orientation of the biotin for the carboxyl transfer, and 12 CoA ester sites. Thus, each of the 6 polypeptides of the central subunit has dual sites for the 3 separate functions. In addition, the two faces of the central subunit apparently have different structures since the dissociation of outer subunits from its two faces is not random; on dissociation, 3 outer subunits are lost from one face prior to loss from the other face. The aims are to determine which portions of the biotinyl subunit are involved in its various functions, determine the sequence of the various sites on the other subunits and relate these to the overall structure and catalysis by this multi subunit enzyme. The long term goal is to gain understanding of how complex enzymes in general are assembled and how the subunits interact in catalysis.